Method for the elimination of kunitz and bowman-birk trypsin inhibitors and carboxypeptidase inhibitor from potato proteins

ABSTRACT

A method for removing protein impurities from extracts of protease inhibitor-containing plant material. Plant materials containing protease inhibitors, such a potato tubers that contain protease inhibitor II, are extracted using an alcohol-free solvent. The proteins present in the extract include impurities other than the protease inhibitor, specifically Kunitz, Bowman-Birk and carboxypeptidase inhibitors. The extract is subjected to heat treatment to denature and precipitate the unstable protein impurities followed by centrifugation to remove the precipitate. Ultrafiltration in the presence of a buffer removes the Bowman-Birk and carboxypeptidase inhibitors. The resulting purified protease inhibitor has applicability in the control of obesity and diabetes.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The invention relates generally to the removal of proteinaseinhibitors from plant sources and, more specifically, to the removal ofKunitz and Bowman-Birk trypsin inhibitors and carboxypeptidaseinhibitors from proteins extracted from potato tubers.

[0003] 2. Background of the Prior Art

[0004] Proteins that inhibit proteolytic enzymes are often found in highconcentrations in many seeds and other plant storage organs. Inhibitorproteins are also found in virtually all animal tissues and fluids.These proteins have been the object of considerable research for manyyears because of their ability to complex with and inhibit proteolyticenzymes from animals and microorganisms. The inhibitors have becomevaluable tools for the study of proteolysis in medicine and biology.Protease inhibitors are of particular interest due to their therapeuticpotentials in controlling proteinases involved in a number of disorderssuch as pancreatitis, shock, and emphysema, and as agents for theregulation of mammalian fertilization. Potato tubers are a rich sourceof a complex group of proteins and polypeptides that potently inhibitseveral proteolytic enzymes usually found in animals and microorganisms.In particular, potato inhibitors are known to inhibit human digestiveproteinases, and thus have application in the control of obesity anddiabetes.

[0005] Proteinase inhibitors found in plants are typically polypeptidesand proteins that are composed entirely of L-amino acids through peptidebonds. These proteinase inhibitors differ significantly in theirproperties. The association of natural proteinase inhibitors with theproteinases that they inhibit is strong at neutral pH, and associationconstants are usually in the range of 10⁷-10¹⁴M⁻¹. Such associations arepH-dependent, and they decrease rapidly as the pH is lowered fromneutrality to 3 (Ryan, C. A., and Walker-Simmons, M. 1981. PlantProteinase. In The Biochemistry of Plants, V6, pp. 321-350, AcademicPress).

[0006] Plant proteinase inhibitors generally are quite stable moleculesand are often resistant to heat, pH extremes, and proteolysis byproteinases, even by those they do not inhibit. This stability has beenattributed in part to the high degree of cross-linking through disulfidebridges. Other, non-covalent interactions also contribute significantlyto the stability of the inhibitors. For example, protease inhibitor Ifrom potatoes is a powerful chemotrypsin inhibitor that, while stable insolution at 80° C. for several minutes (Melville, J. C., and Ryan, C. A.Chemotrypsin inhibitor I from potatoes. J. Microb. Chem. 247: 3445-3453,1972), contains only one disulfide bond per monomer unit (MW ˜8,300)that can be reduced and carboxymethylated without loss of inhibitoryactivity (Plunkett, G., and Ryan, C. A. Reduction andcarboxamidomethylation of the single disulfide bond of proteinaseinhibitor I from potato tubers. Effects on stability, immunologicalproperties, and inhibitory activities. J. Biol. Chem., 255: 2752-2755,1980).

[0007] Several proteinases exhibit substrate specificity, whereasothers, such as papain, have broad substrate specificity. Specificproteinase inhibitor families were identified for each of the fourmechanistic classes of proteolytic enzymes, i.e., serine, cysteinyl,aspartyl and metallo-proteases (Ryan, C. A. Proteinase Inhibitors. InThe Biochemistry of Plants, V6, pp.351-370, Academic Press). In othercircumstances, identical abundant proteins were capable of inhibitingenzymes of various families and have very different substratespecificity, such as the inhibitors of both proteinases and α-amylaseswhich were isolated from cereal seeds (Campos, F. A. P., and Richardson,M. The complete amino acid sequence of α-amylases/trypsin inhibitor fromseeds of ragi (Indian finger millet; Eleusine coracana Goertn.). FEBSLett., 152: 300-304, 1983; Campos, F. A. P., and Richardson, M. Thecomplete amino acid sequence of α-amylases/trypsin inhibitor from seedsof ragi (Indian finger millet; Eleusine coracana Goertn.). FEBS Lett.,167: 221-225, 1984).

[0008] Two broad classes of protease inhibitor superfamilies have beenidentified from soybean and other legumes with each class having severalisoinhibitors. Kunitz-type inhibitor is the major member of the firstclass whose members have 170-200 amino acids, molecular weights between20,000 and 25,000, and act principally against trypsin. Kunitz-typeproteinase inhibitors are mostly single chain polypeptides with 4cysteines linked in two disulfide bridges, and with one reactive sitelocated in a loop defined by disulfide bridge. The second class ofinhibitors contains 60-85 amino acids, has a range in molecular weightof 6000-10,000, has high proportion of disulfide bonds, is relativelyheat-stable, and inhibits both trypsin and chemotrypsin at independentbinding sites. Bowman-Birk inhibitor is an example of this class.

[0009] Kunitz inhibitor is capable of inhibiting trypsin derived from anumber of animal species as well as bovine chemotrypsin, human plasmin,and plasma kallikrein. The cationic form of human trypsin, whichaccounts for a majority of trypsin activity, is only weakly inhibited bythe Kunitz inhibitor, whereas the anionic form is fully inhibited.

[0010] The Bowman-Birk inhibitor is a 71 amino acid chain protein with 7disulfide bonds characterized by its low molecular weight of about 8000(in non-associated monomers), high concentration (about 20%) of cystine,high solubility, resistance to heat denaturation and having the capacityto inhibit trypsin and chymotrypsin at independent inhibitory sites.

[0011] The major effects of proteinase inhibitors in animal dietsinclude growth depression and pancreatic hypertrophy. Resistance of rawsoybean protein to proteolysis, low levels of sulfur-containing aminoacids in soybean proteins, and lower digestibility, absorption, andutilization of available nitrogen from the small intestine due to thepresence of proteinase inhibitors, all appear to contribute to growthdepression.

[0012] Proteinase inhibitors extracted from potatoes have beendistinguished into two groups based on their heat stability. The groupof inhibitors that is stable at 80° C. for 10 minutes have beenidentified as inhibitor I (mol. wt. 39,000) (Melville et al.),carboxypeptidase inhibitor (CPI) (mol. wt. 4,100) (Ryan, C. L.,Purification and properties of a carboxypeptidase inhibitor frompotatoes. J. Biol. Chem. 249: 5495-5499, 1974), inhibitors IIa and IIb(mol. wt. 20,700) (Bryant, J., Green, T. R., Gurusaddaiah, T., Ryan, C.L. Proteinase inhibitor II from potatoes: Isolation and characterizationof its protomer components. Biochemistry 15: 3418-3424, 1976), andinhibitor A5 (mol. wt. 26,000).

[0013] Separation of proteinase inhibitor I by ion exchangechromatography on sulfoethylcellulose in the presence of 0.1 M formicacid in 8 M urea resolved two major and two minor inhibitor protomers.Reassociation by dilution to the tetramer form resulted in two majorprotomers. The first protomer was shown to be a powerful inhibitor ofboth chymotrypsin and trypsin. The second protomer was shown to stronglyinhibit chymotrypsin but only weakly inhibit trypsin. All four purifiedpromoters resolved from Inhibitor I can be reassociated eitherindividually or hybridized with each other to form tetramericisoinhibitors. All of the tetrameric inhibitor I species prepared fromeach of the four protomeric types have glutamic acid at the NH₂terminal. However, they differ from each other in amino acidcomposition, electrophoretic mobility, reactivity with chymotrypsin andtrypsin, and digestibility with pepsin.

[0014] Proteinase inhibitor II, an inhibitor of chemotrypsin andtrypsin, which are serine proteases, is also a heat stable protein. Ithas a dimeric molecular weight of 21,000. Four monomeric isoinhibitorspecies of molecular weight 10,500 comprise inhibitor II and have beenisolated by chromatography in the presence of urea. Upon removal of theurea, each monomeric species dimerized to yield homogenous dimers. Thethree major protomer species, called B, C, and D were found to havesimilar molecular weights and amino acid compositions, and each has anN-terminal alanine residue. Reconstituted dimers possess two bindingsites for bovine α-chymotrypsin, indicating that each monomer possessesone binding site for this enzyme. Significant differences have beennoted among the reconstituted dimers in their isoelectric points,immunoelectrophoretic mobilities, ion-exchange properties, and theirinhibitory reactivities against trypsin. The properties of the inhibitorII dimeric species are similar but not identical to inhibitors IIa andIIb reported from Japanese potatoes, indicating the existence ofintervarietal, as well as intravarietal, differences among potato tuberinhibitor II isoinhibitors (Bryant et al.).

[0015] Protease inhibitor II is composed of two sequence repeats. Itcontains two reactive site domains. The role of the two reactive sitesin the inhibition of trypsin and chemotrypsin has been evaluated. Thefirst reactive site inhibits only chymotrypsin (Ki=2 nM), and thisactivity is very sensitive to mutations. The second reactive sitestrongly inhibits trypsin (Ki=0.4 nM) and chemotrypsin (Ki=0.9 nM), andis quite stable towards mutations (Beekwilder, J., Schipper, B., Bakker,P., Bosch, D., and Jongsma, M. Characterization of potato proteinaseinhibitor II reactive site mutants. Eur. J. Biochem., 267: 1975-1984,2000).

[0016] In addition to inhibitor I and inhibitor II, several lowmolecular weight inhibitors have been detected in potato. Among them arethe carboxypeptidase inhibitor, which has been extensively characterized(Bryant et al., 1976 and Iwasaki, T., Kijohara, T., and Yoshikawa. J.Biol. Chem. (Tokyo) 72: 1029, 1972) and at least three inhibitors ofserine proteinases. The amino acid sequences of two low molecular weightserine proteinase inhibitors from Russet Burbank potatoes have beendetermined. One of those, a chemotrypsin inhibitor, is a peptide of 52amino acid residues, while the second inhibitor, which is specific fortrypsin, contains 51 amino acid residues. These peptides are highlyhomologous, differing at only nine positions. At position 38, thechymotrypsin inhibitor possesses leucine and the trypsine inhibitor anarginine. The inhibitors are also homologous with potato inhibitor IIand with an inhibitor previously isolated from eggplants (Hass, et al.,1982).

[0017] U.S. Pat. No. 5,187,154 describes a method for the diagnosis andthe treatment of individuals with diabetes or at risk to developdiabetes mellitus. In particular, gastric emptying determinations areused to assess risk. Risk or early symptoms associated with subsequentdevelopment of diabetes mellitus may be controlled or alleviated bydelaying gastric emptying, which was achieved by the administration ofcholecystokinin.

[0018] U.S. Pat. No. 4,906,457 describes compositions and methods forreducing the risk of skin cancer. The described compositions included atleast one effective protease inhibitor. Preferred protease inhibitorsincluded serine protease inhibitors and metallo-protease inhibitors. Theprotease inhibitors were preferably included in concentrations rangingfrom approximately 10 picograms to 10 milligrams per milliliter of theskin-applicable topical mixtures. The topical mixtures preferablyincluded a suitable topical vehicle such as a cream, lotion, orointment. One class of anti-carcinogenic skin treatment compositions ofthis invention preferably included the desired protease inhibitors incombination with a suitable sunscreen agent or agents, such aspara-amino benzoic acid, to provide particularly advantageouscompositions for reducing the risk of sunlight-induced skin cancer.

[0019] When applied to mouse epidermal JB6 cells, proteinase inhibitorsI and II from potatoes blocked the UV induced transcription factoractivator protein-1 (AP-1), which has been shown to be responsible forthe tumor promoter action of UV light in mammalian cells. The inhibitionappears to be specific for UV induced signal transduction for AP-1activation. Furthermore, the inhibition of UV induced AP-1 activityoccurs through a pathway that is independent of extracellularsignal-regulated kinases and c-jun N-terminal kinases as well as P38kinases (Huang, C., Ma, W. Y., Ryan, C. A., Dong, Z. Proteinaseinhibitors I and II from potatoes specifically block UV-inducedactivator protein-1 activation through a pathway that is independent ofextracellular signal regulated kinases, c-jun N-terminal kinases, andP38 kinase. Proc. Natl. Acad. Sci., US, 94: 11957-11962, 1997).

[0020] U.S. Pat. No. 4,491,578 describes a method of eliciting satietyin mammals through the administration of an effective amount of atrypsin inhibitor. The method was based on the postulate that the enzymetrypsin, normally secreted by the pancreas, constitutes a negativefeedback signal for cholecystokinin secretion that in turn comprises aputative satiety signal. Thus, the effect of the trypsin inhibitor is toincrease the concentration of cholecystokinin secretion advancing thesensation of satiety resulting in a consequent decrease in food intakeand, over time, body weight.

[0021] The effect of PI2 extracted from potatoes, which increases CCKrelease, or food intake was examined in 11 lean subjects. Five minutesbefore presenting them with a lunchtime test meal, volunteers received1.5 g PI2 in a high protein soup vehicle (70 kcal), the soup vehiclealone, or a no-soup control, according to a double blind, within subjectdesign. The consumption of the soup alone led to a non-significant 3%reduction in energy intake. The addition of 1.5 g PI2 to the soupsignificantly reduced energy intake by additional 17.5%. Pre-mealratings of motivation to eat and food preferences did not predict thereduction in energy intake by the proteinase inhibitor. Based on theresults, the authors concluded that endogenous CCK may be have animportant role in the control of food intake and that proteinaseinhibition may have a potential for reducing food intake (Hill et al.,1990). Clinical trials on potato extracts containing Kunitz inhibitorsshowed no effect on satiety.

[0022] The efficiency of oral trypsin/chemotrypsin inhibitor in delayingthe rate of gastric emptying in recently diagnosed type II diabeticpatients and improving their post-prandial metabolic parameters havebeen examined (Schwartz, J. G., Guan, D., Green, G. M., Phillips, W. T.Treatment with an oral proteinase inhibitor slows gastric emptying andactually reduces glucose and insulin levels after a liquid meal in typeII diabetic patients. Diabetes Care, 17: 255-262, 1994). Serum insulin,plasma glucose, plasma gastric inhibitory polypeptide levels, and therate of gastric emptying were all significantly decreased over the 2hour testing period in subjects who received proteinase inhibitor intheir oral glucose/protein meal. U.S. Pat. No. 5,187,154 suggests theadministration of CCK through an intramuscular injection or anintranasal spray. Alternatively, an oral administration of an agent thatenhances endogenous release of CCK could represent an important approachto the treatment of Type 2 diabetes. One of the agents that may have atherapeutic application in patients with recently diagnosed Type 2diabetes can be the potato proteinase inhibitor II.

[0023] Others have attempted to remove impurity proteins by the use ofchromatography, including ion exchange, gel-filtration and affinity(Mellville et al.), ethanol protein solution (Bryant et al.), andprecipitation with salt and solvents followed by dialysis (Pearce, G.and Ryan, C. A. A rapid, large-scale method for the purification ofmetallo-carboxypeptidase from potato tubers. Anal. Biochem. 30: 223-225,1983). These methods can not be practiced feasibly on a productionscale.

SUMMARY OF THE INVENTION

[0024] The invention consists of a process which utilizes heat treatmentof potato proteins in the presence of salt, followed by centrifugationand filtration, as an efficient method for the elimination of Kunitzfamily, Bowman-Birk proteinase and carboxypeptidase inhibitors fromother potato proteinase inhibitors. Raw potatoes are mixed with anorganic acid, preferably formic acid, and a salt, preferably sodiumchloride. The mixture is comminuted to reduce the size and increase thesurface area of potato particles. The soluble proteins, including PI1,PI2, Kunitz family, Bowman-Birk and carboxypeptidase inhibitors, arereleased into the liquid phase and the mixture is centrifuged to removesolids.

[0025] The supernatant is incubated at a temperature of between about60° C. and about 80° C., and preferably between about 70° C. and 73° C.,for between about 30 minutes and about 180 minutes, and preferablybetween about 45 minutes and 75 minutes, to denature the impurityproteins without denaturing PI2. The solubility of the impurities wasfurther reduced by lowering the temperature of the heat-treated materialto between about 20° C. and about 30° C., and preferably between about25° C. and about 26° C., at which temperatures PI2 remains soluble inthe supernatant.

[0026] Centrifugation for 500,000 g-seconds or longer is used to removethe denatured impurity proteins from the heat-treated supernatant.Ultrafiltration using a cellulosic or sepharose membrane combined withdiafiltration against an ammonium bicarbonate buffer is used to removethe carboxypeptidase inhibitor.

[0027] The process of the present invention is highly efficient in theseparation and removal of Kunitz type inhibitor, previously found tointerfere with the satiety efficacy of the PI2 in humans, as well asBowman-Birk and carboxypeptidase inhibitors. The process also provideshigh recovery and yield of the PI2 inhibitor, increasing theconcentration of PI2 in the final product by more than 100 times incomparison to its concentration in the proteins fraction in the rawpotatoes. The process is efficient at laboratory, pilot plant andproduction scales, is easy to perform and does not require specializedequipment.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028]FIG. 1 is a chromatogram of a Kunitz inhibitor standard made usingreverse phase HPLC.

[0029]FIG. 2 is a chromatogram of a Bowman-Birk inhibitor standard madeusing reverse phase HPLC.

[0030]FIG. 3 is a chromatogram of a carboxypeptidase inhibitor standardmade using reverse phase HPLC.

[0031]FIG. 4 is a chromatogram of the purified PI2 extract of thepresent invention made using reverse phase HPLC under the sameconditions as FIGS. 1-3.

[0032]FIG. 5 is a chromatogram of the purified protease inhibitor IIpeak (i.e., F2) following removal of the Kunitz inhibitors made usingreverse phase HPLC.

[0033]FIG. 6 is a chromatogram of the F2 fraction of FIG. 5 made usinggel filtration HPLC.

[0034]FIG. 7 is a chromatogram of the protein extract after heattreatment and clarification but before filtration.

[0035]FIG. 8 is a chromatogram of the protein extract of FIG. 7 afterfiltration.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

[0036] The efficacy of potato PI2 to function as a satiety aid wasstudied in a clinical trial in which the PI2 had a protein purity of notless than 50%. Unfortunately, extracting PI2 from potatoes results inthe concomitant extraction of impurities, including proteins such asKunitz, Bowman-Birk, and carboxypeptidase inhibitors. Since certain ofthese impurities are believed to interfere with the efficacy of PI2, aprocess for their removal is needed.

[0037] PI2 is known to have significant heat stability in comparisonwith certain other soluble proteins found in potatoes. Through the useof heat treatment, protein impurities that are less heat stable can beeliminated from the liquid phase by heating the extract to a temperatureand for a time period sufficient to denature the non-stable proteinswithout substantially denaturing the PI2, followed by removal of thedenatured proteins. An experiment was conducted to examine the maximumtemperature of the heat-treatment step, the hold-time at the elevatedtemperature, and a cooling temperature that was expected to reduce thesolubility of the denatured proteins so that they can be removed fromthe solution. At the same time, loss of soluble PI2, removal of solublenon-PI2 proteins, and precipitation of insoluble materials wereexamined. The majority of the undesirable, non-stable proteins arebelieved to be Kunitz type proteins. In using the high-performanceliquid chromatography (HPLC) methodology applicable for PI2quantification, the Kunitz type impurities are those proteins whichelute following the target PI2 protein and the carboxypeptidaseinhibitor.

[0038] The carboxypeptidase inhibitor (CPI) is shown to haveheat-stability similar to PI2 as observed by the limited loss of thisimpurity during the heat-treatment step. While the heat stability of theBowman-Birk impurities is not well-characterized, it has been assumedthat some or all may not be denatured during the heat treatment step andso, like the carboxypeptidase inhibitor, must be removed by a differentmethod. Given the difference in molecular weight between the Bowman-Birk(approx. 8000 Daltons) and carboxypeptidase inhibitor (approx. 4100Daltons) compared to PI2 (approx. 21,700 Daltons), molecular size-basedseparation techniques were investigated.

[0039] Extraction and Purification Process

[0040] The extraction and isolation of PI2 from potatoes begins with theaddition of an organic acid, preferably formic acid, and a salt,preferably sodium chloride, to raw potatoes. The mixture is subjected tocomminution to increase the surface area of the potato particles andimprove the extraction of soluble proteins. Centrifugation is used toremove solids, and the liquid fraction is heated at a temperaturesufficient to denature many undesired proteins but not PI2. The solutionis again centrifuged to remove the insoluble denatured proteins and theliquid fraction is microfiltered to remove relatively large particles.Ultrafiltration is used to further purify the PI2 in the retentate.

[0041] More specifically, whole, raw potatoes are added to an aqueoussolvent containing formic acid and sodium chloride. The potatoes andsolvent are comminuted to reduce the potato particle size and increasethe exposed surface area to improve extraction efficiencies. A filtercentrifuge is used to remove bulk fiber and starch while retaining thesoluble proteins in the liquid fraction. The filtrate is heated to lessthan 80° C. for between about 30 minutes and 3 hours and then cooled toapproximately 25° C. A tubular bowl clarifier is used to remove thenon-heat stable impurities, notably the Kunitz inhibitors. A microfilter(0.3 m) is used to remove particles not removed by the clarifier.Ultrafiltration using a filter having a molecular weight cutoff ofbetween approximately 5000 and 10,000 Daltons in the presence of adiafiltration buffer, preferably ammonium bicarbonate, is used to removeimpurities generally smaller than the molecular weight cutoff, notablythe Bowman-Birk and carboxypeptidase inhibitors. The retentate containspurified PI2 and may be lyophilized to generate a dried product.

[0042] Even more specifically, whole, raw potatoes, preferably thosehaving a high PI2 content, are added to an extractant solution orsolvent, comprising water with a sodium chloride content of between 0.3N and 2.0 N to which is added between 0.5 and 2.5 weight percent formicacid, at a weight ratio of between 1:1 and 1:10 raw potatoes to solvent,and preferably about 1:2.5. A grind profile which results in an averageparticle size of approximately 500 m is used to create a relativelyfinely comminuted product without unduly heating of the slurry. Thefilter centrifuge uses a 35 m bag mesh set at approximately 75% load.Heat is applied to bring the liquid extract to a temperature ofapproximately 70° C. for approximately one hour. Cooling the extract tobetween 20° C. and 26° C. causes precipitation of the denatured proteinswhile the PI2 remains in solution. Centrifugation at approximately13,000×g removes the denatured proteins. The clarifier is operated toreduce the weight percent of solids to about 0.01% or below. Acellulosic, open, screen channel membrane with a molecular weight cutoffof approximately 10,000 Daltons is used at a flow rate of 0.40 litersper minute per square foot of membrane surface, with a 20 psi pressuredifferential. Six volumes of 100 mM ammonium bicarbonate buffer are usedin the diafiltration.

[0043] Reverse Phase HPLC Method

[0044] The amount of PI2, Kunitz and carboxypeptidase inhibitors wasmeasured using reverse phase HPLC. A Microsorb C-18 column (4.6 mm×250mm, 5 μm particles with 300 Angstrom pore size; Varian AnalyticalInstruments) was used. Two mobile phase solvents were prepared, solventA was 800 g deionized H₂O, 150 g acetonitrile, and 0.95 trifluoroaceticacid, and solvent B was 850 g acetonitrile and 0.85 g trifluoroaceticacid. Approximately 50 mg of the sample was added to 100 ml of solventA. The sample was vortexed for 30 seconds, and then centrifuged at10,000 rpm for 10 minutes. The supernatant was collected for RP-HPLCanalysis. One hundred μl of the sample was injected into the column,with the pump set at 800-2500 psig, and a temperature of 30.0° C. Theother flow rate, time, and solvent compositions are as set out inTable 1. The diode array of the detector was set at 220 nm. TABLE 1 HPLCConditions Solvent Composition Time (min) Flow rate (ml/min) (volume %) 0 1 100% A  5 1 100% A 34 1  38% A 38 1 100% B 40 2 100% B 45 2 100% B50 1 100% A 55 1 100% A

[0045] An external standard was prepared to construct a standard curvefor calibration of the column. Five mg of BSA were dissolved in 10 ml ofsolvent A. Four volumes, i.e., 25, 50, 75, and 100 μL, were injectedinto the column. A calibration curve was generated from the results.

[0046] Gel Filtration Chromatography HPLC for the Purification of PI2

[0047] A method was developed to separate PI2 from other heat stablepotato proteins using gel filtration high-performance liquidchromatography. A Shodex protein KW-803 column (8 mm×300 mm, 5 μmparticles with 120 Angstrom pore size) was used. An isocratic 0.025 Mphosphate buffer was used, and the flow rate was set to 0.25 ml/min, andthe pressure mainained at 40 bar. The diode array of the detector wasset at 220 nm. A 1 L volume of a 0.025 M potassium phosphate buffer, pH7.0, was prepared for use as the mobile phase by adding 2.67 g K₂HPO₄and 1.31 g of K₂H₂PO₄ to 900 ml of deionized water. After verificationof a pH of 7.0, or adjustment to 7.0 using either HCl or NaOH,additional water is added to bring the volume to 1 L. The following HPLCgradient was used to flush the 5 ml manual injector sample loop. TABLE 2HPLC Conditions Time (min) Flow rate (ml/mm) 0 0.5 10 0.5 12 0.25

[0048] Kunitz and Carboxypeptidase Standards

[0049] To establish the removal of Kunitz and carboxypeptidaseimpurities from the potato extract, the reverse phase HPLC method wasused on commercially available Kunitz and carboxypeptidase standards.Both standards were purchased from SIGMA. A chromatogram of the Kunitzstandard is illustrated in FIG. 1 and a chromatogram of thecarboxypeptidase standard is illustrated in FIG. 3. Note that the majorpeak of the Kunitz impurities appears at 24.4 minutes, and the majorpeaks of the carboxypeptidase impurities appear at 17.4-17.8 and 18.9minutes.

[0050] Bowman-Birk Standard

[0051] A commercially available Bowman-Birk standard was purchased fromSIGMA and analyzed using the reverse phase HPLC method. A chromatogramof the Bowman-Birk standard is illustrated in FIG. 2. Note that themajor peaks occur at 13 and 14.8 minutes.

[0052] Gel Electrophoresis Method

[0053] Gel electrophoresis used to analyze samples described in thisapplication refers to the method described in “Separation Technique FileNo. 110, the Amersham Pharmacia LKB PhastSystem.

[0054] Trypsin Inhibition Assay

[0055] To further support the HPLC results indicating the presence ofPI2, trypsin inhibition was applied as a biomarker for the efficiency ofthe extraction of PI2. A method was developed to assay the amount oftrypsin inhibition demonstrated by the extracts of the presentinvention. The materials used included Trizma pre-set crystals, Trypsin(Worthington Biochemical LS003703), Nα-p-Tosyl-L-arginine methyl ester(TAME)(Sigma T-4626), and Trypsin Inhibitor (Sigma catalog #T-9003).

[0056] A hydrochloric acid solution, (0.001N) was prepared by mixing 81μl of concentrated HCl and 1L of deionized H₂O. Tris buffer was preparedby mixing 1.74 g of Trizma pre-set crystals (pH 8.3) and 0.42 g calciumchloride in 250 ml of DI H₂O. Trypsin stock solution was prepared bydissolving 7.5 mg of trypsin in 5ml of 0.001 N HCl. The concentration oftrypsin in the solution was determined by measuring the absorbance at280 nm.

[Trypsin](mg/ml)=A₂₈₀*0.70

[0057] Twenty μl of the trypsin stock solution were added to 1.98 ml of0.001N HCl to make the trypsin working solution. Trypsin solutions werekept on ice. A trypsin solution was freshly prepared after the 5^(th) or6^(th) sample set if more than 6 samples were to be analyzed, since thetrypsin activity diminishes over the period of time needed for analysis.TAME (38mg) was dissolved in 10 ml 0.001 N HCl. This solution wasfreshly made on a daily basis.

[0058] A PI2 stock solution was prepared by dissolving 35.0 mg of samplein 10 ml of DI H₂O. This sample was also used for the protein assay. Inaddition, a 3.5 mg trypsin inhibitor (TI) sample (Sigma catalog #T-9003)was prepared in 10 ml DI H₂O. The sample was vortexed until completelydissolved, and then centrifuged for 10 min at 10,000 rpm. Thesupernatant served as the PI-2 or TI stock solution used for thepreparation of PI2 or TI working solutions (Tables 3 and 4).

[0059] The following PI2 working solutions were prepared: TABLE 3Working Solution PI2 stock solution (μl) 0.001 N HCl (μl) A  25 1975 B 50 1950 C  75 1925 D 100 1900 E 300 1700 F 500 1500

[0060] The following TI std working solutions were prepared: TABLE 4Working Solution TI stock solution (μl) 0.001 N HCl (μl) A  25 1975 B 50 1950 C  75 1925 D 100 1900 E 300 1700 F 500 1500

[0061] The following solutions were prepared for UV/VIS analysis:

PI2 Samples:

[0062] TABLE 5 Buffer Solution X solution Trypsin working 0.001 N HClSample (μl) (μl) solution (μl) (μl) 1-Blank 0 200  0 200  2-Uninhibited0 200 100 100  3 A(100) 200 100 0 4 B(100) 200 100 0 5 C(100) 200 100 06 D(100) 200 100 0 7 E(100) 200 100 0 8 F(100) 200 100 0

TI Standards:

[0063] TABLE 6 Buffer Solution X solution Trypsin working 0.001 N HClSample (μl) (μl) solution (μl) (μl) 1-Blank 0 200  0 200  2-Uninhibited0 200 100 100  3 A(100) 200 100 0 4 B(100) 200 100 0 5 C(100) 200 100 06 D(100) 200 100 0 7 E(100) 200 100 0 8 F(100) 200 100 0

[0064] The samples were allowed to equilibrate for 15 min. Afterequilibration, 2.3 ml of buffer solution were added. Just prior toreading samples, 300μl of TAME solution were added. The sample was thenvortexed and immediately transferred to a cuvette (1 cm pathlength). Thesamples were read at 247 nm at 10 second intervals for 3 minutes, andthe reported inhibition rate (AU/min) was recorded. Negative controlsconsisting of TI standards or PI2 stock solution that have been heatedfor 1 hour at 110° C. can be included, but are not necessary. Previouswork has shown that in comparison to buffer solutions, these inactivatedprotein solutions have no trypsin inhibition.

[0065] The following calculations are used:

[0066] 1) Net uninhibited rate (δA₂₄₇/min)=Uninhibited rate−Blank rate

[0067] 2) Net inhibited rate (δA₂₄₇/min)=Inhibited rate−Blank rate

[0068] 3) Trypsin in reaction mix(mg)=Trypsin stock conc(mg/ml)/1000

[0069] 4) Lyophilized solid (μg)=Solid(mg)*PI2 stock added to work sol'n(ml)*5

[0070] 5) PI2 (μg)=(Solid(mg)*PI2 stock added to work sol'n(ml)* %purity)/20

[0071] 6)${{Trypsin}\quad {activity}\quad \text{(}{units}\text{/}{mg}\quad {enzyme}\text{)}} = \frac{{Net}\quad {rate}{\quad \quad}\text{(}\Delta \quad {A_{247}/\min}\text{)}*1000\quad*3}{540*{trypsin}\quad {in}\quad {rxn}\quad {mixture}\quad ({mg})}$

[0072] (The extinction coefficient of Nα-p-Tosyl-L-arginine=540(“Trypsinogen-Trypsin.” Ed. Charles C. Worthington. Worthington EnzymeManual. Freehold, N.J.: Worthington Biochemical Corporation, 1988.320-324))

[0073] 7) Net Remaining Trypsin Activity in each sample or standard(units)=Remaining Activity−Activity in Blank

[0074] 8) Plots of trypsin activity vs. PI2 and/or lyophilized solids,proteins or PI2 (mg) were constructed. The point on the graph at whichthe activity has been reduced by ˜50% is selected. The determinedactivity at this point and the amount of lyophilized solid, proteins orPI2 in the reaction mixture can be used to express the results as

[0075] mg Trypsin Inhibited/mg Lyophilized Solid

[0076] mg Trypsin Inhibited/mg Proteins

[0077] mg Trypsin Inhibited/mg PI2

[0078] according to the following formulas:

[0079] 8a) mg Trypsin Inhibited=(Net Total Uninhibited Activity−NetRemaining Uninhibited Activity)*trypsin in rxn mixture (mg)

[0080] 8b)${{mg}\quad {Trypsin}\quad {Inhibited}\text{/}{mg}\quad {Lyophilized}\quad {Solid}} = \frac{{{mg}\quad {Trypsin}\quad {Inhibited}*1000\quad {\mu g}\text{/}{mg}}}{{\mu g}\quad {solid}\quad {in}\quad {rxn}\quad {mixture}}$

[0081] 8c)${{mg}\quad {Trypsin}\quad {Inhibited}\text{/}{mg}\quad {PI2}} = \frac{{mg}\quad {trypsin}\quad {inhibited}\quad*1000\quad {\mu g}\text{/}{mg}}{{\mu g}\quad {PI2}\quad {in}\quad {rxn}\quad {mixture}}$

[0082] In this assay a trypsin inhibitor unit was defined as the amountof the trypsin inhibitor that caused inhibition of 50% of the 0.2865units of trypsin used in the reaction mixture, under the conditionsapplied for the reaction. This value will be taken from the standardinhibitor/inhibition curve. In the end, results were reported as unitsof trypsin inhibition/ mg solids or proteins.

[0083] Experiment 1

[0084] The temperature required to denature and precipitate proteinimpurities was first examined. A lot of potatoes was extracted andfiltered using an extractant consisting of 1.0 N sodium chloride and1.5% formic acid. This extract was filtered and aliquoted for theheat-treatment study. Each sample of the extract was placed in a testtube, and then heated to the target temperature using a constanttemperature water bath. Samples were taken at times 0 minutes, 15minutes, 30 minutes, 45 minutes and 60 minutes. The samples wereimmersed in an ice bath and then centrifuged in an Eppendorf 5415centrifuge for 5 minutes at 10,000 rpm to remove precipitated material.The supernatant was analyzed using the reverse phase HPLC methoddescribed above and the Kunitz peaks were quantified. The results arereported in Table 7. TABLE 7 Kunitz Impurities Remaining in SolutionTemperature ° C. Time (minutes) Kunitz¹ mg/ml 70 0 0.819 70 15 0.158 7030 0.128 70 45 0.119 70 60 0.110 80 0 0.874 80 15 0.131 80 30 0.105 8045 0.101 80 60 0.095 90 0 0.878 90 15 0.112 90 30 0.108 90 45 0.100 9060 0.099

[0085] It is clear from the data that there is a time advantage to begained, in terms of rapidity of impurity removal, by treating theproduct at a temperature of 90° C. After 15 minutes at 90° C. theremoval of the Kunitz type proteins was equivalent to that of heatingfor 60 minutes at 70° C. or heating for 30 minutes at 80° C.Unfortunately, as PI-2 has limited stability at 90° C., it is necessaryto treat at a lower temperature to minimize PI2 degradation and loss.Removal of the Kunitz type proteins was efficiently accomplished byincreasing the product temperature to 70° C. for 60 minutes. Heating theextract to 70° C. for 15 minutes generated an 81% reduction in theamount of Kunitz proteins and after 60 minutes at 70 ° C. this reductionhad reached of 87%.

[0086] A further trial was run to determine the effect of heat treatmentat various temperatures and time periods on the amount of PI2,carboxypeptidase inhibitor (CPI), Kunitz type proteins and overallpurity. The conditions and results are as reported in Table 8. TABLE 8Protein purities of temperature trials from 60° C. through 80° C. SamplePI2 (mg/ml) CPI (mg/ml) Kunitz (mg/ml) Overall purity PI2/Kunitz PurityHT 60° C. 0.27 0.22 1.32 14.70% 16.75%  0 min HT 60° C. 0.26 0.22 0.7720.74% 25.28% 15 min HT 60° C. 0.25 0.22 0.64 22.61% 28.18% 30 min HT70° C. 0.21 0.19 0.29 29.93% 41.58% 15 min HT 70° C. 0.21 0.20 0.1338.18% 61.49% 30 min HT 80° C. 0.24 0.22 0.19 36.82% 55.35% 15 min HT80° C. 0.24 0.22 0.26 33.48% 48.25% 30 min

[0087] Data in Table 8 provide supporting evidence of the use of aproduct temperature of 70° C. to maximize product purity. Of particularinterest is the marked reduction in purity associated with the datataken at 60° C. This can possibly be explained by the incompleteprecipitation of the Kunitz impurities below 70° C.

[0088] Experiment 2

[0089] Trials were conducted to determine the selection of anultrafiltration system and operating conditions which would be effectiveat removing the Bowman-Birk and carboxypeptidase inhibitors withoutremoving an excess of PI2. Three different filter types were examined,the Pall Filtron Centramate CS010C12, (Pall Corporation, East Hills,N.Y.), the Pall Filtron Maximate CS010G02, and the A/G TechnologyUFP-5-C-4A, (A/G Technology Corporation, Needham, Mass.). Each filterwas tested under a range of conditions consistent with itsspecifications. All of the filters were found to be non-fouling underthe tested conditions, but the Maximate filter had an average flux of 63liters/hour/meter², compared to 54.7 for the Centramate and 20.2 for theA/G Technology filter.

[0090] Relative recovery of the PI2 after ultrafiltration was examined.While the A/G Technology membrane is rated at a molecular cutoff of 5000Daltons and the Pall Filtron membrane is rated at 10,000 Daltons, thepercentage of PI2 recovery was 7.60 for the A/G Technologies membraneand 9.61 for the Poll Filtron membrane. It is believed that differencesin pore geometries of the Pall Filtron cellulosic membrane and the A/GTechnolgy polysulfone membrane result in differences in filtration thatare dependent on molecular geometry in addition to molecular weight.Tests using a membrane having a molecular cutoff of 1000 Daltonsresulted in a dramatic reduction in flux.

[0091] Diafiltration against water resulted in rapid and irreversiblefouling of the membrane with a corresponding flux rate decay. The use ofa 100 mM ammonium bicarbonate buffer during the diafiltration phaseprevented fouling of the membrane and allowed for the removal of theBowman-Birk and carboxypeptidase inhibitors. Integration of the HPLCchromatographs prior to and after ultrafiltration/diafiltrationdemonstrated that the heat stable impurities were present at aboutone-third of the concentration of PI2. Ultrafiltration did notsignificantly change the ratio of impurity to PI2. Diafiltration against6 volumes of 100 mM ammonium bicarbonate results in a reduction of theratio of impurity to PI2 of greater than 50% (Table 9). Diafiltrationagainst larger volumes of ammonium bicarbonate exhibited almost completeremoval of the impurities after 20 volumes. The ratio of impurities toPI2 was observed to remain essentially unchanged when using water inplace of the filtration buffer. TABLE 9 Effect of Diafiltration with 100mmol Ammonium Bicarbonate Doublet Impurity PI2 Integrated IntegratedRatio Impurity: Sample Area (mAU) Area (mAU) PI2 Heat Treated 2917 11190.384 Extract Concentrated 17133 6574 0.384 Extract  6X Diafiltered25166 3767 0.150 against AMBI 10X Diafiltered 13967 1135 0.081 againstAMBI 20X Diafiltered 17965 281 0.016 against AMBI ˜5X Diafiltered 46481596 0.343 against Water

[0092] The heat treatment step denatures the Kunitz impurities and theyare precipitated and removed by centrifugation. The carboxypeptidaseinhibitor is known to have a heat stability similar to that of PI2 andso is not believed to be substantially denatured during the heattreatment step. While the heat stability of the Bowman-Birk impuritiesis not well-characterized, it may be assumed that some may be denaturedand precipitated during the heat treatment step, but others may remainin the extract after centrifugation. Protease inhibitor II has amolecular weight of approx. 20,700 Daltons, Bowman-Birk have a molecularweight of approx. 8000 Daltons, and carboxypeptidase inhibitor has amolecular weight of approx. 4100 Daltons.

[0093] Six samples of PI2 extracted from potato tubers following themethod described above were prepared. Each of the samples was separatedusing the reverse phase-HPLC method described above, with the followingmodifications: (a) the column used was a Microsorb C-4 (otherwiseunchanged); (b) solvent A comprised 900 g DI H₂O and 0.90trifluoroacetic acid; and (c) the solvent composition was 80% A in thefirst and last 10 minute periods and 30% A in the 10 to 15 minuteperiod. In each separation, three fractions were collected and retained,referred to as the fractions F1, F2, and F3 of FIG. 5, taken at timeintervals of 17-32.5 minutes, 32.5-36 minutes, and 36-46 minutes,respectively. Note the carboxypeptidase inhibitor doublet at 36-38minutes.

[0094] The volume of each F2 sample was reduced to 500 1 using aRoto-Vap under reduced pressure. Each of the samples was then separatedusing gel filtration-HPLC according to the previously described method.In each separation, three fractions were collected and retained,referred to as fractions F1, F2, and F3 of FIG. 5, taken at timeintervals of 10-29.6 minutes, 29.6-34.5 minutes, and 34.5-70 minutes,respectively.

[0095] Following reverse phase chromatography of a sample using theprevious procedure, the PI2 F2 peak (FIG. 5) was collected. The samplewas reduced to approximately 100 μl using a Rotovap and deionized waterwas added to bring the volume to 500 μl. A manual injector was used toinject the sample into the GFC column. The chromatogram following gelfiltration HPLC is illustrated in FIG. 6 (note that the peak at 36minutes is an artifact). Expressing the TI results vs. PI2 proteincontent based on the GFC peak area showed an increase of ˜35 times inthe specific activity of the PI2 (Table 10). TABLE 10 Trypsin activityexhibited by various PI2 preparations expressed by various means TIActivity ( /mg) Sample Solids PI2 in F2 Actual PI2 (GFC) 1/22/01UF 76.341960.78 384.62 bulk batch #1 54.05 1960.78 476.19 1/12/01UF 32.261428.57 322.58 12/12/01UF 76.34 3846.15 526.32 12/9/01UF 31.75 2127.66322.58 12/29/01UF 74.07 1000.00 210.53 average 57.47 2053.99 373.80 stddev 21.43  973.31 114.69 range: 31.75-76.34  1428.57- 210.53-526.323846.15

[0096] Trypsin inhibition activity was detected in all of the threepeaks separated by GFC-HPLC. However the inhibition activity detected inthe early and late eluting peaks was less than the inhibition levelsdetected in the GFC PI2 peak. Once present together, proteins eluting inother peaks such as those eluting in peak 1 and peak 3 may work insynergy with proteins from GFC PI2 peak. Variability among inhibitionresults was still observed in the GFC PI2 product. However, as can bedepicted from the trypsin inhibitor activity content shown in Table 10,this variability was less than what was observed by expressing theresults vs. solids or calculated PI2 content based on the GFC peak area.The trypsin inhibition activity content in the GFC PI2 peak was about20% of the content determined based on calculated PI2 content in thesame peak (Table 10). This is probably due to elution of trypsininhibition activity that is not necessarily due to PI2, under the earlyand late appearing peaks separated by RP-HPLC (FIG. 5) and/or GFC-HPLC.This interpretation is partially supported by the fact that variousprotein levels were detected in the three peaks separated by GFC-HPLC(Table 11). TABLE 11 Protein content in various PI2 product fractionsTotal Bradford Protein Bradford based based content actual actual actualtotal total in GFC proteins in proteins in proteins in solids proteinpeaks GFC F1 GFC F2 GFC F3 (mg) (mg)* (mcg) (mcg) (mcg)** (mcg)  1/225.25 0.743 195.8 40.2 118.6 37.2 (14.13%) (21.1%) (60.6%) (20%) bulk5.25 0.704 155.7 38.1 63.5 54.6 batch (13.42%) (24.5%) (40.8%) (35.1%)#1  1/12 5.25 0.698 140.3 35.5 86.7 18.1 (13.30%) (25.3%) (61.8) (12.9%)12/12 5.25 0.961 149.9 47.3 66.5 36.1 (18.30%) (31.6%) (44.4%) (24.1%)12/9  5.25 0.803 213   60.6 108.1 44.3 (15.29%) (28.5%) (50.8%) (20.8%)12/29 5.25 0.760 205.2 53.6 92.5 59.1 (14.48%) (26.1%) (26.1%) (28.8)

[0097] Experiment 3

[0098] To verify that the peak in the F2 region of FIG. 5 containspotato PI2, a sample of PI2 was obtained from Dr. Clarence Ryan atWashington State University. The sample of PI2 was prepared by Dr.Ryan's laboratory following the method described in Melville et al. ThePI2 standard and a sample of the purified PI2 extract produced using themethod of this specification were analyzed using the gel electrophoresismethod described above. Also run on the gel were molecular weightstandards, including—Lactalbumin (MW=14,400) and soybean trypsininhibitor (MW=20,100). The PI2 standard showed a band in agreement witha band of the PI2 sample of the present invention. Moreover, there wereno bands prior to the -Lactalbumin standard marker band at 14,400Daltons. Accordingly, the gel electrophoresis results demonstrate thatthe F2 region of FIG. 5 includes the PI2 extracted from the potatoes.

[0099] To verify the removal of the impurities, the reverse phase HPLCmethod was used to analyze a sample of the purified PI2 extract. Achromatogram of the PI2 sample is illustrated in FIG. 4. The principalpeak at 15.4 minutes is the peak containing the PI2. Based on theanalysis of the carboxypeptidase standard (FIG. 3) and the Kunitzstandard (FIG. 1), very little of the impurities show up on the HPLCchromatogram of FIG. 4, except for a small amount of thecarboxypeptidase doublet that can be seen at approximately 18 minutes,and a small amount of the Kunitz peak that can be seen at approximately25 minutes. The effect of filtration on the removal of the CPI isillustrated in the two HPLC chromatograms of FIGS. 7 and 8. The peak atapproximately 15 minutes is the PI2 peak, with the CPI doublet appearingat 17-18 minutes. The dramatic reduction in the size of the CPI doubletis clearly shown by the relative changes in the PI2 peak and the CPIdoublet.

[0100] As demonstrated in FIG. 2, the Bowman-Birk standard elutes underreverse phase HPLC at approximately the same time as the PI2 peak ofFIG. 4. Accordingly, HPLC could not be used, as with Kunitz andcarboxypeptidase, to demonstrate removal of Bowman-Birk impurities.Note, however, that the gel electrophoresis pattern reported aboveshowed no bands smaller than 14,400 Daltons. Since the Bowman-Birkimpurities are known to have a molecular weight of approximately 8,000Daltons, the gel electrophoresis results demonstrate removal of theBowman-Birk impurities. Further, since the carboxypeptidase impuritieshave a molecular weight of 4,100 Daltons, the gel electrophoresisresults also support the removal of the carboxypeptidase impurities.

[0101] The foregoing description and drawings comprise illustrativeembodiments of the present inventions. The foregoing embodiments and themethods described herein may vary based on the ability, experience, andpreference of those skilled in the art. Merely listing the steps of themethod in a certain order does not constitute any limitation on theorder of the steps of the method. The foregoing description and drawingsmerely explain and illustrate the invention, and the invention is notlimited thereto, except insofar as the claim are so limited. Thoseskilled in the art who have the disclosure before them will be able tomake modifications and variations therein without departing from thescope of the invention.

We claim:
 1. A method of removing Kunitz, Bowman-Birk andcarboxypeptidase inhibitors from an extract of proteaseinhibitor-containing plant material using an alcohol-free, aqueoussolvent, comprising the steps of: (a) heating the extract to atemperature and for a period of time to denature a substantial amount ofthe Kunitz inhibitors without denaturing a substantial amount of theprotease inhibitor; (b) cooling the extract to reduce the solubility ofthe denatured proteins; (c) removing the denatured proteins from thecooled extract to leave a purified extract; and (d) filtering thepurified extract to remove the carboxypeptidase and Bowman-Birkinhibitors.
 2. The method of claim 1, wherein the plant material ispotato tubers.
 3. The method of claim 1, wherein the protease inhibitoris protease inhibitor II.
 4. The method of claim 1, wherein thedenatured proteins are removed by centrifuigation.
 5. The method ofclaim 1, wherein the filtering step is carried out in the presence of abuffer.
 6. The method of claim 5, wherein the buffer is ammoniumbicarbonate.
 7. The method of claim 1, wherein the filtering stepcomprises utltrafiltration.
 8. The method of claim 1, wherein theheating step is carried out at a temperature between about 65° C. andabout 90° C. for between about 15 minutes and about 180 minutes, andwherein the cooling step reduces the temperature of the extract tobetween about 20° C. and about 30° C.
 9. A method of removing Kunitz,Bowman-Birk and carboxypeptidase inhibitors from proteaseinhibitor-containing plant material, comprising the steps of: (a)extracting the plant material using an alcohol-free, aqueous solvent toform an extract containing the protease inhibitor; (b) heating theextract to a temperature between about 65° C. and about 90° C. forbetween about 15 minutes and about 180 minutes to denature the Kunitzinhibitors; (b) cooling the extract to between about 20° C. and about30° C.; (c) centrifuging the cooled extract to remove the denaturedKunitz inhibitors and form a clarified extract; (d) usingultrafiltration in the presence of a buffer to remove the Bowman-Birkand carboxypeptidase inhibitors from the clarified extract.
 10. Themethod of claim 9, wherein the solvent comprises formic acid and sodiumchloride.
 11. The method of claim 9, wherein the buffer comprisesammonium bicarbonate.
 12. The method of claim 9, wherein the heatingtemperature is between about 68° C. and about 73° C. and the heatingtime is between about 45 and about 75 minutes.
 13. The method of claim9, wherein the cooling step reduces the temperature of the extract tobetween about 20° C. and about 26° C.